Recombination between sequences on two distinct duplex DNAs, or between sequences, which are separated by an intervening sequence on the same DNA duplex, occurs in all cells capable of self-replication. Four-way Holliday junctions in DNA are a feature of recombination reactions and are generated in both homologous and site-specific recombination reactions (Lilley, D. M. J. PNAS 94, 9513-9515 (1997)). The penultimate stage of recombination involves resolution of the four-way junction by catalysis. The catalysis is achieved by structure-specific nucleases identified as resolvases (Aravind, L., et al. Nuc. Acid Res. 28:3417-3432 (2000)). Resolvases are widespread in cells and additionally are expressed by viruses. Resolvases have diverse properties. The crystal structures of a number of resolvases have been reported, including that of T7 Endonuclease I (T7 Endo I) (Hadden, J. M., et al. Nat. Struct. Biol. 8:62-67 (2001)).
T7 Endo I has two catalytic centers that are juxtaposed in a way that prevents the enzyme from forming a productive complex with regular linear DNA but enables it to specifically bind and cleave branched, perturbed or flexible DNA. The three-dimensional structure of T7 Endo I shows that the two catalytic domains are well separated and connected only by the β-sheet bridge (Hadden, J. M., et al. Nat. Struct. Biol. 8:62-67 (2001)). The bridge forms part of an extended and tightly associated anti-parallel β-sheet (β2).
T7 Endo I is a stable homodimer of 149 amino acid subunits (Parkinson, M. J. and Lilley, D. M. J. J. Mol. Biol. 270:169-178 (1997)). It is very basic (pI calc 9.5) and binds tightly (Kd 2nM) to four-way junctions in dimeric form. T7 Endo I resolves four-way junctions by simultaneously introducing two nicks on the two continuous strands, at sites 5′ to the junction (Déclais, A., et al. EMBO J. 22:1398-1409 (2003)). This structural analysis shows that T7 Endo I forms an intimately associated symmetrical homodimer comprising two catalytic domains connected by a bridge. Each catalytic domain is composed of residues 17-44 from one subunit and residues 50-145 from the other.
Resolvases including T7 Endo I are structure-specific endonucleases capable of cleaving a broad range of DNA molecules with a variety of structures such as branched structures and single-base mismatched heteroduplexes (Mashal, R. D., et al. Nat. Genet. 9:177-183 (1995)). This broad substrate specificity makes it difficult to identify the common structural features of the substrate that the enzyme selectively recognizes and cleaves (White, M. F., et al. J. Mol. Biol 269:647-664 (1997)). The broad substrate specificity may also contribute to the toxicity of the resolvase in host cells.
It would be desirable to reduce the toxicity of the enzymes having a similar structure to T7 Endo I to facilitate their overproduction for use as reagents in molecular biology. It would also be desirable to selectively favor specific enzyme activities.